Biological and Terahertz-Derived Signatures of Polymer Nanocarrier-Induced Cellular Responses: Toward Structure–Response Relationships in Photodynamic Therapy

Abstract Polymer micelles based on poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-PCL) are widely investigated as nanocarriers for photodynamic therapy (PDT) due to their ability to solubilize hydrophobic photosensitizers such as pheophorbide a and enhance their delivery. In this study, we systematically assessed the impact of the morphology (spherical PEO5000-PCL4000 micelles vs PEO2000-PCL7000 worm-like nanovectors) and crosslinking status on PDT responses in both 2D cell cultures and 3D spheroid models. Although all pheophorbide-loaded formulations produced comparable phototoxicity in normal human dermal fibroblasts, human HCT-116 tumor cells, and canine MDCK-1 epithelial cells, detailed mechanistic analyses revealed distinct early cellular responses. Viability assays, membrane integrity measurements, ion homeostasis analyses, scanning electron microscopy, and terahertz (THz) spectroscopy demonstrated rapid membrane permeabilization, ionic imbalance, and ultrastructural remodeling following PDT. Worm-like nanovectors induced more pronounced membrane vesiculation, while THz spectroscopy revealed formulation-dependent membrane dynamics. Unexpectedly, blank nanovectors also altered ionic homeostasis and cellular behavior, indicating that these carriers are not biologically inert. Multivariate analyses integrating biological and THz-derived physical parameters generated distinctive signatures of nanocarrier formulations, enabling discrimination according to cargo loading, morphology, and crosslinking status. Together, these findings establish a multidimensional framework for characterizing nanocarrier effects on cells and highlight the value of combining biological and biophysical measurements to guide the rational design of PDT nanomedicines.